GSM6589210: HCT116, MLL4 KO, MLL4; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
KMT2D
Cell type
Cell type Class
Digestive tract
Cell type
HCT 116
Primary Tissue
Colon
Tissue Diagnosis
Carcinoma
Attributes by original data submitter
Sample
source_name
HCT116
cell line
HCT116
cell type
Human colorectal cancer
genotype
MLL4 KO
chip antibody
MLL4 (homemade, #3)
Sequenced DNA Library
library_name
GSM6589210
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were cross-linked with 1% formaldehyde for 10 min. Crosslinking reaction was stopped by 125 mM glycine for 10 min. 30 – 40 million cross-linked cells were resuspended in 5 mL lysis buffer (5 mM PIPES, pH 7.5, 85 mM KCl, 1% NP-40 and protease inhibitors), incubated on ice for 10 min, and spun down at 1,000 g for 5 min at 4°C. The pellet was washed once with 5 mL lysis buffer. Resulting nuclei were resuspended with 2 mL TE buffer (50 mM Tris-HCl, pH8.0, 10 mM EDTA and protease inhibitors) and sonicated for 10 min (30 sec on and 30 sec off) at 20% amplitude. After adding detergents to a final concentration of 1% Triton-X100, 0.1% SDS, 0.1% sodium deoxycholate (1x RIPA), sonicated chromatin was spun down at 15,000 g for 10 min at 4°C. For each ChIP, 10 µg of target antibodies and 2 μg of spike-in antibody (anti-H2Av, Active Motif, #61686) were mixed with 400 – 600 mg sonicated chromatin and incubated on a rotator at 4°C overnight. Next day, 50 μL prewashed protein A Dynabeads (Thermo Fisher) were added to chromatin-antibody complex and incubated for 2 hours at 4°C. ChIPed samples were washed twice with 1 mL RIPA, twice with 1 mL RIPA + 300mM NaCl, twice with 1 mL LiCl buffer and once with PBS. Samples were eluted in 100 μL buffer containing 0.1M NaHCO3, 1% SDS, and 20μg Proteinase K at 65°C overnight and DNA was purified using QIAquick PCR purification kit (Qiagen). Libraries were prepared according to NEB (Illumina kit) instructions. DNA were used to construct libraries using NEBNext® Ultra™ II DNA Library Prep kit with AMPure XP magnetic beads (Beckman Coulter).